Amorphous solid form of a bet protein inhibitor

ABSTRACT

The present invention relates to an amorphous solid form of (4S)-7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(M)-one, and processes for its preparation, which is an inhibitor of BET proteins such as BRD2, BRD3, BRD4, and BRD-t and is useful in the treatment of various diseases such as cancer.

FIELD OF THE INVENTION

The present invention relates to an amorphous solid form of(4S)-7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one,and processes for its preparation, which is an inhibitor of BET proteinssuch as BRD2, BRD3, BRD4, and BRD-t and is useful in the treatment ofvarious diseases such as cancer.

BACKGROUND OF THE INVENTION

The BET (Bromodomain and Extra-Terminal) family of bromodomaincontaining proteins comprises 4 proteins (BRD2, BRD3, BRD4 and BRD-t)that share a conserved structural organization containing tandemN-terminal bromodomains capable of binding to acetylated lysine residuesof histones and other proteins. BRD2, BRD3 and BRD4 are ubiquitouslyexpressed while BRD-t is restricted to germ cells. BRD proteins playessential, but non-overlapping roles in regulating gene transcriptionand controlling cell growth. BET proteins are associated with largeprotein complexes including Mediator, PAFc and super elongation complexthat regulate many aspects of gene transcription. BRD2 and BRD4 proteinshave been shown to remain in complex with chromosomes during mitosis andare required to promote transcription of critical genes including cyclinD and c-Myc that initiate the cell cycle. Mochizuki et al., J. Biol.Chem. 2008, 283, 9040-9048. BRD4 is essential for recruiting the proteintranslational elongation factor B complex to the promoters of induciblegenes resulting in the phosphorylation of RNA polymerase II andstimulating productive gene transcription and elongation. Jang et al.,Mol. Cell, 2005, 19, 523-534. In some instances, a kinase activity ofBRD4 may directly phosphorylate and activate RNA polymerase II. Devaiahet al., Proc. Nat. Acad. Sci., USA. 2012, 109, 6927-6932. Cells lackingBRD4 show impaired progression through cell cycle. BRD2 and BRD3 arereported to associate with histones along actively transcribed genes andmay be involved in facilitating transcriptional elongation. Leroy etal., Mol. Cell, 2008, 30, 51-60. In addition to acetylated histones, BETproteins have been shown to bind selectively to acetylated transcriptionfactors including the RelA subunit of NF-kB and GATA1 thereby directlyregulating the transcriptional activity of these proteins to controlexpression of genes involved in inflammation and hematopoieticdifferentiation. Huang et al., Mol. Cell Biol., 2009, 29, 1375-1387;Lamonica et al., Proc. Nat. Acad. Sci., USA, 2011, 108, E159-168.

A recurrent translocation involving NUT (nuclear protein in testes) withBRD3 or BRD4 to form a novel fusion oncogene, BRD-NUT, is found in ahighly malignant form of epithelial neoplasia. French et al., CancerRes., 2003, 63, 304-307; French et al., J. Clin. Oncol., 2004, 22,4135-4139. Selective ablation of this oncogene restores normal cellulardifferentiation and reverses the tumorigenic phenotype. Filippakopouloset al., Nature, 2010, 468, 1068-1073. Genetic knockdown of BRD2, BRD3and BRD4 has been shown to impair the growth and viability of a widerange of hematological and solid tumor cells. Zuber et al., Nature,2011, 478, 524-528; Delmore et al., Cell, 2011, 146, 904-917. Aside froma role in cancer, BET proteins regulate inflammatory responses tobacterial challenge, and a BRD2 hypomorph mouse model showeddramatically lower levels of inflammatory cytokines and protection fromobesity induced diabetes. Wang et al., Biochem. J., 2009, 425, 71-83;Belkina et al., J. Immunol. 102838, online publication before print,Feb. 18, 2013. In addition, some viruses make use of these BET proteinsto tether their genomes to the host cell chromatin, as part of theprocess of viral replication or use BET proteins to facilitate viralgene transcription and repression. You et al., Cell, 2004, 117, 349-60;Zhu et al., Cell Reports, 2012, 2, 807-816.

Inhibitors of BET proteins are in current development. Exemplary BETprotein inhibitors are disclosed in, for example, U.S. Pat. App. Pub.Nos. 2014/0275030; 2015/0011540; 2015/0148375; 2015/0148342;2015/0148372; and 2015/0175604. An example of BET protein inhibitor is(4S)-7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(M)-onewhich is described in U.S. Pat. App. Pub. No. 2014/0275030. Whilecertain inhibitors of BET proteins are in the literature, there remainsa need for new solid forms of these inhibitors having suitableproperties useful in the manufacture of safe, effective, high qualitydrug products. The present invention described herein is directed towardthis end.

SUMMARY OF THE INVENTION

The present invention provides, inter alia, a solid form, which is anamorphous powder, of the BET protein-inhibiting compound(4S)-7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one,compositions, methods of use, and methods for preparing the same.

The present invention also provides intermediate compounds generatedduring the preparation of(4S)-7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-oneand methods for preparing these intermediate compounds.

The details of one or more embodiments are set forth in the descriptionbelow. Other features, objects, and advantages will be apparent from thedescription and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an XRPD pattern for Compound 1-(S) amorphous powder.

FIG. 2 shows a DSC thermogram for Compound 1-(S) amorphous powder.

FIG. 3 shows a MIDSC thermogram for Compound 1-(S) amorphous powder.

DETAILED DESCRIPTION

Solid Form

The present invention provides, inter alia, a solid form, which is anamorphous solid, of the BET protein-inhibiting compound(4S)-7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(M)-one(see below), referred to herein as “Compound 1-(S).” In one embodiment,the solid is an amorphous powder. An alternative name for the samecompound is(3S)-6-(3,5-dimethylisoxazol-4-yl)-3-(pyridin-2-yl)-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-2(1H)-one.

The amorphous solid form of the invention has several properties makingit particularly suitable for scale up and formulation. For example, thesolid form of the invention has good stability with respect todecomposition and unwanted conversion to crystalline forms as evidencedby stability studies (see Example 5) and a relatively high glasstransition temperature (see Example 4). Additionally, the amorphoussolid form can be reliably prepared in high purity, with little to nounwanted residual organic solvent. Residual organic solvent can includeany solvents that were employed during the synthesis of the solid form,or residues or impurities present in the starting materials andreagents.

In some embodiments, the solid form of the invention is characterized byan XRPD (X-ray power diffraction) pattern substantially as shown inFIG. 1. As can be seen in the XRPD pattern, no reflections are observed,indicating a homogenous amorphous solid form. The XRPD is obtained frompowder diffractometer with X-ray radiation from copper at 1.054056 Åwith K_(β) filter and X-ray power at 30 KV, 15 mA, and the sample powderwas dispersed on a zero-background sample holder (see Example 2).

In some embodiments, the solid form of the invention is characterized bya DSC (differential scanning calorimetry) thermogram substantially asshown in FIG. 2. As can be seen in FIG. 2, the DSC is characterized byan exothermic peak at about 213° C. which is believed to correspond to adecomposition event. In some embodiments, the DSC is characterized by anexothermic peak with an onset at about 182° C. The DSC is obtained fromTA Instruments Differential Scanning calorimetry, Model Q200 withautosampler (see Example 3).

In some embodiments, the solid form of the invention has a glasstransition temperature (Tg) of about 106° C. The glass transitiontemperature is determined by modulated DSC (MDSC) using TA InstrumentsDifferential Scanning calorimetry, Model Q2000 with autosampler.

The solid form of the invention can be prepared in high purity. Purityvalues presented herein indicate the percentage of the amount of samplethat is e.g., Compound 1-(S) (including stereoisomers thereof). Purityvalues can be determined, for example, by HPLC/UV methods. In someembodiments, the amorphous form of the invention has a purity greaterthan about 90%, greater than about 95%, greater than about 97%, greaterthan about 98%, greater than about 98.5%, or greater than about 99%. Insome embodiments, the amorphous form of the invention is substantiallyfree of impurities, such as decomposition products and/or residualorganic solvent. In some embodiments, the primary impurity in theamorphous form of the invention is water, which can be present, forexample, in an amount of less than about 5%, less than about 3%, lessthan about 2%, less, than about 1.5%, less than about 1%, or less thanabout 0.5%.

The solid form of the invention can be made by initially dissolvingCompound 1-(S) in water with the aid of a base (e.g., a strong base).While not wishing to be bound by theory, it is believed that the base(e.g., strong base) assists dissolution of Compound 1-(S) bydeprotonation of the weakly acidic cyclic urea group. In this way, asufficient amount of Compound 1-(S) can be dissolved in aqueous solutionfor purification. As used herein, the term “aqueous solution” refers toa solvent system comprised primarily of water. In some embodiments, theaqueous solution is free from organic substances, such as organicsolvents, with the exception of Compound 1-(S). In some embodiments, theaqueous solution may contain acids and/or bases, such as inorganic acidsand/or bases. Additionally, the aqueous solution may contain one or moresalts or ions, such as inorganic salts or ions.

After the Compound 1-(S) is dissolved, the highly basic solution isacidified, which reduces the solubility of Compound 1-(S) and causes itto precipitate out of the aqueous solution into a non-crystalline,homogenous powder which is amenable to use in the formulation of drugproducts. This process does not require the use of any organic solvents,which is advantageous with respect to scale up and environmentalconcerns, as well as resulting in a drug product substantially free frompotentially harmful organic residue.

In some embodiments, the present invention provides a method forpreparing(4S)-7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(M)-one(Compound 1-(S)) in the form of an amorphous powder comprisingprecipitating Compound 1-(S) from an aqueous solution comprisingCompound 1-(S).

The aqueous solution is initially basic, having a pH greater than 7,such as from about 10-14, about 11-13, or about 12-13. The aqueoussolution can be made basic by the addition of base, such as a strongbase. Examples of bases include NaOH, KOH, LiOH, and CsOH. In someembodiments, the base is NaOH. Precipitation of Compound 1-(S) iscarried out by acidification of the basic aqueous solution in whichCompound 1-(S) is dissolved. The acidification can be carried out byaddition of acid, such as a strong acid. Examples of acids include HCl,HBr, H₂SO₄, H₃PO₄, methanesulfonic acid (CH₃SO₃H), and toluenesulfonicacid (pTsOH). In some embodiments, the acid is HCl. Acidification mayresult in an aqueous solution having a final pH below 7, such as a pHabout 1-5, about 1-4, or about 2-4.

In some embodiments, the present invention provides a method forpreparing(4S)-7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(M)-one(Compound 1-(S)) in the form of an amorphous powder, comprising:

a) dissolving Compound 1-(S) in a solvent system comprising water and abase (e.g., a strong base) to form a basic aqueous solution;

b) adding an acid (e.g., a strong acid) to the basic aqueous solution(e.g., in an amount effective to lower the pH below about 7) toprecipitate the Compound 1-(S) as an amorphous powder.

In one embodiment, the step of adding converts the basic solution to anacidic solution having a pH below about 7.

In some embodiments, the base comprises NaOH.

In some embodiments, the solvent system comprising water and a base(e.g., a strong base) is substantially free of organic solvent.

In some embodiments, the solvent system comprising water and a base(e.g., a strong base) is substantially free of any organic molecules,except for Compound 1-(S) and stereoisomers thereof.

In some embodiments, the basic aqueous solution has a pH of about 10-14.

In some embodiments, the basic aqueous solution has a pH of about 12-13.

In some embodiments, the basic aqueous solution is filtered prior toadding an acid (e.g., a strong acid).

In some embodiments, the acid comprises HCl.

In some embodiments, adding the acid lowers the pH of the aqueoussolution to a pH of about 1-5

In some embodiments, adding the acid lowers the pH of the aqueoussolution to a pH of about 2-4.

In some embodiments, the present invention provides a solid form ofCompound 1-(S) which is prepared by any of the methods described herein.

The present invention also provides a compound, wherein the compound is5-nitro-3-(pyridin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-3-ol(Compound 1x) or a salt thereof (e.g., a pharmaceutically acceptablesalt thereof). Compound 1x or a salt thereof can be prepared in highpurity. Purity values can be determined, for example, by HPLC/UVmethods. In some embodiments, Compound 1x or a salt thereof has a puritygreater than about 90%, greater than about 95%, greater than about 97%,greater than about 98%, or greater than about 99%.

The present invention also provides a compound, wherein the compound is7-iodo-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 4x) or a salt thereof (e.g., a pharmaceutically acceptablesalt thereof). Compound 4x or a salt thereof can be prepared in highpurity. Purity values can be determined, for example, by HPLC/UVmethods. In some embodiments, Compound 4x or a salt thereof has a puritygreater than about 90%, greater than about 95%, greater than about 97%,greater than about 98%, or greater than about 99%.

In some embodiment, the present invention also provides a method forpreparing Compound 1-(S), comprising:

reacting7-iodo-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 4x) with (3,5-dimethylisoxazol-4-yl)boronic acid to afford7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 1) in the presence of a palladium complex; and separating theS enantiomer of Compound 1 using chiral column chromatography to affordCompound 1-(S).

The present invention also provides a method for preparing Compound1-(S), comprising the steps of:

1) reacting 2-bromo-1-pyridin-2-ylethanone hydrobromide with2-amino-nitrophenol in the presence of a base and an organic solvent toafford 5-nitro-3-(pyridin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-3-ol(Compound 1x);

2) reacting Compound 1x with hydrogen in the presence of palladium oncarbon and an organic solvent to afford3-(pyridin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-5-amine (Compound2x);

3) reacting Compound 2x with N,N-carbonyldiimidazole in the presence ofan organic solvent to afford4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 3x);

4) reacting Compound 3x with N-iodosuccinimide to afford7-iodo-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 4x);

5) reacting Compound 4x with (3,5-dimethylisoxazol-4-yl)boronic acid inthe presence of a palladium complex to afford7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 1); and

6) separating the S enantiomer of Compound 1 through chiral columnchromatography to afford Compound 1-(S).

In step 1, 2-bromo-1-pyridin-2-ylethanone hydrobromide is reacted with2-amino-nitrophenol in the presence of a base and an organic solvent toafford 5-nitro-3-(pyridin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-3-ol(Compound 1x). In some embodiments, the present invention also providesa method for preparing5-nitro-3-(pyridin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-3-ol(Compound 1x), comprising reacting 2-bromo-1-pyridin-2-ylethanonehydrobromide with 2-amino-nitrophenol in the presence of a base and anorganic solvent. In some embodiments, the base is K₂CO₃. In someembodiments, the organic solvent is acetonitrile.

In step 2, Compound 1x is reacted with hydrogen in the presence ofpalladium on carbon and an organic solvent to afford Compound 2x. Insome embodiments, the solvent is methanol.

In step 3, Compound 2x is reacted with N,N-carbonyldiimidazole in thepresence of an organic solvent to afford Compound 3x. In someembodiments, the organic solvent is ethyl acetate.

In step 4, Compound 3x is reacted with N-iodosuccinimide (NIS) to affordCompound 4x. In some embodiments, the present invention provides amethod for preparing7-iodo-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 4x), comprising reacting4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 3x) with N-iodosuccinimide (NIS). In some embodiments, thereacting is carried out in the presence of an organic solvent and anacid. In some embodiments, the organic solvent isN,N-dimethyl-acetamide. In some embodiments, the acid is sulfuric acid.In some embodiments, the amount of sulfuric acid is about 0.1-0.5 molarequivalent of Compound 3x. In some embodiments, the amount of sulfuricacid is about 0.1, about 0.2, about 0.3, about 0.4, or about 0.5 molarequivalent of Compound 3x. In some embodiments, the amount of sulfuricacid is about 0.3 molar equivalent of Compound 3x.

Using MS in step 4 provides certain advantages over other halogenationreagents, for example, N-bromosuccinimide (NBS). While not wishing to bebound by theory, NIS is believed to improve the regioselectivity of theresulting product. For example, if NB S is used in this electrophilicbromination reaction, the ratio of the desired (para-) regioisomer tothe undesired (ortho-) regioisomer is about 5 to 1. By comparison, usingMS in step 4 has an improved ratio of about 10 to 1. It is believed thatbecause NBS is more reactive than NIS, NBS generally provides a lowerregioselectivity towards electrophilic halogenation reaction on theactivated aromatic substrate. Another advantage of using NIS is that theresulting aryliodide compound provides a faster and clean Suzukicoupling reaction in the subsequent step as compared to e.g., thecorresponding arylbromo compound.

In step 5, Compound 4x is reacted with(3,5-dimethylisoxazol-4-yl)boronic acid in the presence of a palladiumcomplex to afford Compound 1. In some embodiments, the present inventionprovides a method for preparing7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 1), comprising reacting7-iodo-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 4x) with (3,5-dimethylisoxazol-4-yl)boronic acid in thepresence of a palladium complex. In some embodiments, the reacting iscarried out in the presence of an aqueous CsF solution and an organicsolvent. In some embodiments, the amount of palladium complex is about0.001-0.010 molar equivalent of Compound 4x. In some embodiments, theamount of palladium complex is about 0.001, about 0.002, about 0.003,about 0.004, about 0.005, about 0.006, about 0.007, about 0.008, about0.009, or about 0.010 molar equivalent of Compound 4x. In someembodiments, the palladium complex is dichlorobis(p-dimethylaminophenylditbutylphosphine)palladium(II). In some embodiments, the organicsolvent is n-butanol.

Using (3,5-dimethylisoxazol-4-yl)boronic acid instead of thecorresponding pinacol ester has certain advantages, especially on largescale processes. For example, boronic acid is usually more reactive thanits pinacol ester and thus, less reaction time;(3,5-dimethylisoxazole)boronic acid is commercially available andeconomically less expensive; the boronic acid is a white solid which iseasier to handle (e.g., shipping and charging, etc.).

In step 6, Compound 1 is purified through chiral column chromatographyto afford Compound 1-(S). Compound 1 in acetonitrile is loaded onto thecolumn, which is eluted with acetonitrile. The chiral purity of thefractions containing Compound 1-(S) is determined by chiral HPLC. Afterremoval of acetonitrile from the fraction containing Compound 1-(S) toafford a crude Compound 1-(S), the sample is further purified bydissolving in methanol and loaded onto the same chiral column, which iseluted with methanol. The desired fraction containing Compound 1-(S) iscollected and the solvent is removed under reduced pressure to affordCompound 1-(S).

As used herein, the term “organic solvent” refers to carbon-basedsolvents (i.e., they contain carbon in their structure) that areemployed to dissolve or disperse one or more compounds described herein.Examples of organic solvents include but not limited to acetone, aceticacid, acetonitrile, benzene, carbon tetrachloride, chloroform, dimethylsulfoxide, methanol, methyl t-butyl ether, methylene chloride,N,N-dimethylformamide, pentane, ethanol, ethyl acetate, hexanes,isopropanol, tetrahydrofuran, and toluene.

As used herein, the term “solvent system” refers to a system whichcomprises one or more solvents. The system may also contain one or morereagents or starting materials used to prepare a particular compound. Insome embodiments, the system may contain primary of water or organicsolvents. The system may also contain acids and/or bases, such asinorganic acids and/or bases.

Compounds described herein also include tautomeric forms. Tautomericforms result from the swapping of a single bond with an adjacent doublebond together with the concomitant migration of a proton. Tautomericforms include prototropic tautomers which are isomeric protonationstates having the same empirical formula and total charge. Exampleprototropic tautomers include ketone—enol pairs, amide—imidic acidpairs, lactam—lactim pairs, amide—imidic acid pairs, enamine—iminepairs, and annular forms where a proton can occupy two or more positionsof a heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H-and 4H-1, 2, 4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

Compounds described herein can also include all isotopes of atomsoccurring in the intermediates or final compounds. Isotopes includethose atoms having the same atomic number but different mass numbers.Compounds of the invention can also include all isotopes of atomsoccurring in the intermediates or final compounds. Isotopes includethose atoms having the same atomic number but different mass numbers.For example, isotopes of hydrogen include tritium and deuterium. One ormore constituent atoms of the compounds of the invention can be replacedor substituted with isotopes of the atoms in natural or non-naturalabundance. In some embodiments, the compound includes at least onedeuterium atom. For example, one or more hydrogen atoms in a compound ofthe present disclosure can be replaced or substituted by deuterium. Insome embodiments, the compound includes two or more deuterium atoms. Insome embodiments, the compound includes 1, 2, 3, 4, 5, 6, 7 or 8deuterium atoms. Synthetic methods for including isotopes into organiccompounds are known in the art.

Substitution with heavier isotopes such as deuterium, may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample, increased in vivo half-life or reduced dosage requirements, andhence may be preferred in some circumstances. (A. Kerekes et. al. J.Med. Chem. 2011, 54, 201-210; R. Xu et. al. J. Label Compd. Radiopharm.2015, 58, 308-312).

The term, “compound,” as used herein is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted. Compounds herein identified by name or structure asone particular tautomeric form are intended to include other tautomericforms unless otherwise specified (e.g., in the case of purine rings,unless otherwise indicated, when the compound name or structure has the9H tautomer, it is understood that the 7H tautomer is also encompassed).

The expressions, “ambient temperature” and “room temperature,” as usedherein, are understood in the art, and refer generally to a temperature,e.g., a reaction temperature, that is about the temperature of the roomin which the reaction is carried out, for example, a temperature fromabout 20° C. to about 30° C.

Temperature values in connection with DSC, TGA, or other thermalexperiments can vary about ±3° C. depending on the instrument,particular settings, sample preparation, etc. Accordingly, a solid formreported herein having a DSC thermogram “substantially” as shown in anyof the Figures is understood to accommodate such variation.Additionally, the recitation of temperature values together with theterm “about” for peaks or other events in connection with a thermogramalso accommodates such variation.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The present invention also includes salts (e.g., pharmaceuticallyacceptable salts) of the compounds described herein. As used herein,“salts” refers to derivatives of the disclosed compounds wherein theparent compound is modified by converting an existing acid or basemoiety to its salt form. Examples of salts include, but are not limitedto, mineral or organic acid salts of basic residues such as amines;alkali or organic salts of acidic residues such as carboxylic acids; andthe like. The salts of the present invention include the conventionalnon-toxic salts of the parent compound formed, for example, fromnon-toxic inorganic or organic acids. The salts of the present inventioncan be synthesized from the parent compound which contains a basic oracidic moiety by conventional chemical methods. Generally, such saltscan be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g.,methanol, ethanol, iso-propanol, or butanol) or acetonitrile (MeCN) arepreferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th) Ed., (Mack Publishing Company, Easton,1985), p. 1418, Berge et al., J. Pharm. Sci., 1977, 66(1), 1-19, and inStahl et al., Handbook of Pharmaceutical Salts: Properties, Selection,and Use, (Wiley, 2002). In some embodiments, the compounds describedherein include the N-oxide forms.

The following abbreviations may be used herein: AcOH (acetic acid); Ac₂O(acetic anhydride); aq. (aqueous); atm. (atmosphere(s)); Boc(t-butoxycarbonyl); br (broad); Cbz (carboxybenzyl); calc. (calculated);d (doublet); dd (doublet of doublets); DCM (dichloromethane); DEAD(diethyl azodicarboxylate); DIAD (N,N′-diisopropyl azidodicarboxylate);DIPEA (N,N-diisopropylethylamine); DMF (N,N-dimethylformamide); Et(ethyl); EtOAc (ethyl acetate); g (gram(s)); h (hour(s)); HATU(N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate); HCl (hydrochloric acid); HPLC (high performanceliquid chromatography); Hz (hertz); J (coupling constant); LCMS (liquidchromatography—mass spectrometry); m (multiplet); M (molar); mCPBA(3-chloroperoxybenzoic acid); MgSO₄ (magnesium sulfate); MS (Massspectrometry); Me (methyl); MeCN (acetonitrile); MeOH (methanol); mg(milligram(s)); min. (minutes(s)); mL (milliliter(s)); mmol(millimole(s)); N (normal); NaHCO₃ (sodium bicarbonate); NaOH (sodiumhydroxide); Na₂SO₄ (sodium sulfate); NH₄Cl (ammonium chloride); NH₄OH(ammonium hydroxide); nM (nanomolar); NMR (nuclear magnetic resonancespectroscopy); OTf (trifluoromethanesulfonate); Pd (palladium); Ph(phenyl); pM (picomolar); POCl₃ (phosphoryl chloride); RP-HPLC (reversephase high performance liquid chromatography); s (singlet); t (tripletor tertiary); TBS (tert-butyldimethylsilyl); tert (tertiary); tt(triplet of triplets); t-Bu (tert-butyl); TFA (trifluoroacetic acid);THF (tetrahydrofuran); μg (microgram(s)); μL (microliter(s)); μM(micromolar); wt % (weight percent).

Methods of Use

Compound 1-(S) is a BET protein inhibitor and, thus, is useful intreating and/or preventing diseases and disorders associated withactivity of BET proteins. For example, Compound 1-(S) can inhibit one ormore of BET proteins BRD2, BRD3, BRD4, and BRD-t. In some embodiments,Compound 1-(S) selectively inhibits one or more BET proteins overanother. “Selective” means that the compound binds to or inhibits a BETprotein with greater affinity or potency, respectively, compared to areference, such as another BET protein.

The solid form of Compound 1-(S) described herein is therefore usefulfor treating and/or preventing BET-mediated disorders. The term“BET-mediated disorder” refers to any disease or condition in which oneor more of the BET proteins, such as BRD2, BRD3, BRD4 and/or BRD-t, or amutant thereof, plays a role, or where the disease or condition isassociated with expression or activity of one or more of the BETproteins. The solid form of Compound 1-(S) described herein can be usedto treat or lessen the severity of diseases and conditions where BETproteins, such as BRD2, BRD3, BRD4, and/or BRD-t, or a mutant thereof,are known to play a role.

Diseases and conditions treatable and/or preventable using the solidform provided herein include cancer and other proliferative disorders,autoimmune disease, chronic inflammatory diseases, acute inflammatorydiseases, sepsis, and viral infection. The diseases can be treated byadministering to an individual (e.g., a patient) in need of thetreatment a therapeutically effective amount or dose of a solid form ofCompound 1-(S) described herein, or any of the embodiments thereof, or apharmaceutical composition thereof.

In some embodiments, the present invention provides a method ofinhibiting a BET protein comprising contacting the BET protein with thesolid form provided herein.

In some embodiments, the present invention provides a method of treatingcancer in a patient, comprising administering to the patient atherapeutically effective amount of the solid form provided herein. Thecancers can include adrenal cancer, acinic cell carcinoma, acousticneuroma, acral lentiginous melanoma, acrospiroma, acute eosinophilicleukemia, acute erythroid leukemia, acute lymphoblastic leukemia, acutemegakaryoblastic leukemia, acute monocytic leukemia, acute promyelocyticleukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoidodontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm,adrenocortical carcinoma, adult T-cell leukemia/lymphoma, aggressiveNK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma,alveolar soft part sarcoma, ameloblastic fibroma, anaplastic large celllymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma,angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoidtumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocyticleukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer,bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor,Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma insitu, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma,chondroma, chordoma, choriocarcinoma, choroid plexus papilloma,clear-cell sarcoma of the kidney, craniopharyngioma, cutaneous T-celllymphoma, cervical cancer, colorectal cancer, Degos disease,desmoplastic small round cell tumor, diffuse large B-cell lymphoma,dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonalcarcinoma, endocrine gland neoplasm, endodermal sinus tumor,enteropathy-associated T-cell lymphoma, esophageal cancer, fetus infetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroidcancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor,gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumorof the bone, glial tumor, glioblastoma multiforme, glioma, gliomatosiscerebri, glucagonoma, gonadoblastoma, granulosa cell tumor,gynandroblastoma, gallbladder cancer, gastric cancer, hairy cellleukemia, hemangioblastoma, head and neck cancer, hemangiopericytoma,hematological malignancy, hepatoblastoma, hepatosplenic T-cell lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma,intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna,lethal midline carcinoma, leukemia, leydig cell tumor, liposarcoma, lungcancer, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma,acute lymphocytic leukemia, acute myelogenous leukemia, chroniclymphocytic leukemia, liver cancer, small cell lung cancer, non-smallcell lung cancer, MALT lymphoma, malignant fibrous histiocytoma,malignant peripheral nerve sheath tumor, malignant triton tumor, mantlecell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia,mediastinal germ cell tumor, medullary carcinoma of the breast,medullary thyroid cancer, medulloblastoma, melanoma, meningioma, merkelcell cancer, mesothelioma, metastatic urothelial carcinoma, mixedMullerian tumor, mucinous tumor, multiple myeloma, muscle tissueneoplasm, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma,nasopharyngeal carcinoma, neurinoma, neuroblastoma, neurofibroma,neuroma, nodular melanoma, ocular cancer, oligoastrocytoma,oligodendroglioma, oncocytoma, optic nerve sheath meningioma, opticnerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor,papillary thyroid cancer, paraganglioma, pinealoblastoma, pineocytoma,pituicytoma, pituitary adenoma, pituitary tumor, plasmacytoma,polyembryoma, precursor T-lymphoblastic lymphoma, primary centralnervous system lymphoma, primary effusion lymphoma, primary peritonealcancer, prostate cancer, pancreatic cancer, pharyngeal cancer,pseudomyxoma peritonei, renal cell carcinoma, renal medullary carcinoma,retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation,rectal cancer, sarcoma, Schwannomatosis, seminoma, Sertoli cell tumor,sex cord-gonadal stromal tumor, signet ring cell carcinoma, skin cancer,small blue round cell tumors, small cell carcinoma, soft tissue sarcoma,somatostatinoma, soot wart, spinal tumor, splenic marginal zonelymphoma, squamous cell carcinoma, synovial sarcoma, Sezary's disease,small intestine cancer, squamous carcinoma, stomach cancer, T-celllymphoma, testicular cancer, thecoma, thyroid cancer, transitional cellcarcinoma, throat cancer, urachal cancer, urogenital cancer, urothelialcarcinoma, uveal melanoma, uterine cancer, verrucous carcinoma, visualpathway glioma, vulvar cancer, vaginal cancer, Waldenstrom'smacroglobulinemia, Warthin's tumor, and Wilms' tumor. In someembodiments, the cancer can be adenocarcinoma, adult T-cellleukemia/lymphoma, bladder cancer, blastoma, bone cancer, breast cancer,brain cancer, carcinoma, myeloid sarcoma, cervical cancer, colorectalcancer, esophageal cancer, gastrointestinal cancer, glioblastomamultiforme, glioma, gallbladder cancer, gastric cancer, head and neckcancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, intestinal cancer,kidney cancer, laryngeal cancer, leukemia, lung cancer, lymphoma, livercancer, small cell lung cancer, non-small cell lung cancer,mesothelioma, multiple myeloma, ocular cancer, optic nerve tumor, oralcancer, ovarian cancer, pituitary tumor, primary central nervous systemlymphoma, prostate cancer, pancreatic cancer, pharyngeal cancer, renalcell carcinoma, rectal cancer, sarcoma, skin cancer, spinal tumor, smallintestine cancer, stomach cancer, T-cell lymphoma, testicular cancer,thyroid cancer, throat cancer, urogenital cancer, urothelial carcinoma,uterine cancer, vaginal cancer, or Wilms' tumor.

In some embodiments, the present invention provides a method of treatinga solid tumor in a patient, comprising administering to the patient atherapeutically effective amount of the solid form provided herein.

In some embodiments, the present invention provides a method of treatingcolorectal cancer, lung cancer, pancreatic cancer, prostate cancer, orbreast cancer in a patient, comprising administering to the patient atherapeutically effective amount of the solid form provided herein.

In some embodiments, the present invention provides a method of treatinglymphoma in a patient, comprising administering to the patient atherapeutically effective amount of the solid form provided herein. Insome embodiments, the lymphoma is diffuse large B-cell lymphoma (DLBCL).

In some embodiments, the present invention provides a method of treatingleukemia in a patient, comprising administering to the patient atherapeutically effective amount of the solid form provided herein. Insome embodiments, the leukemia is acute myeloid leukemia (AML), chronicmyeloid leukemia (CIVIL), atypical chronic myeloid leukemia (aCML), orchronic myelomonocytic leukemia (CMML).

In some embodiments, the present invention provides a method of treatingmyelodysplastic syndrome (MDS), myelodysplastic/myeloproliferativeneoplasms (MDS/MPN), myelofibrosis (MF), multiple myeloma (MM), orrefractory anemia with ringed sideroblasts associated with markedthrombocytosis (RARS-T) in a patient, comprising administering to thepatient a therapeutically effective amount of the solid form providedherein.

In some embodiments, the present invention provides a method of treatingNUT midline carcinoma in a patient, comprising administering to thepatient a therapeutically effective amount of the solid form providedherein.

The diseases treatable using the solid form provided herein also includeMYC dependent cancers wherein the cancer is associated with at least oneof myc RNA expression or MYC protein expression. A patient can beidentified for such treatment by determining myc RNA expression or MYCprotein expression in the cancerous tissue or cells.

Diseases that can be treated with the solid form provided herein alsoinclude non-cancerous proliferative disorders. Examples of proliferativedisorders that can be treated include, but are not limited to, benignsoft tissue tumors, bone tumors, brain and spinal tumors, eyelid andorbital tumors, granuloma, lipoma, meningioma, multiple endocrineneoplasia, nasal polyps, pituitary tumors, prolactinoma, pseudotumorcerebri, seborrheic keratoses, stomach polyps, thyroid nodules, cysticneoplasms of the pancreas, hemangiomas, vocal cord nodules, polyps, andcysts, Castleman disease, chronic pilonidal disease, dermatofibroma,pilar cyst, pyogenic granuloma, and juvenile polyposis syndrome.

The diseases and conditions that can be treated with the solid formprovided herein also include chronic autoimmune and inflammatoryconditions. Examples of autoimmune and inflammatory conditions that canbe treated include acute, hyperacute or chronic rejection oftransplanted organs, acute gout, acute inflammatory responses (such asacute respiratory distress syndrome and ischemia/reperfusion injury),Addison's disease, agammaglobulinemia, allergic rhinitis, allergy,alopecia, Alzheimer's disease, appendicitis, atherosclerosis, asthma,osteoarthritis, juvenile arthritis, psoriatic arthritis, rheumatoidarthriti, satopic dermatitis, autoimmune alopecia, autoimmune hemolyticand thrombocytopenic states, autoimmune hypopituitarism, autoimmunepolyglandular disease, Behcet's disease, bullous skin diseases,cholecystitis, chronic idiopathic thrombocytopenic purpura, chronicobstructive pulmonary disease (COPD), cirrhosis, degenerative jointdisease, depression, dermatitis, dermatomyositis, eczema, enteritis,encephalitis, gastritis glomerulonephritis, giant cell arteritis,Goodpasture's syndrome, Guillain-Barre syndrome, gingivitis, Graves'disease, Hashimoto's thyroiditis, hepatitis, hypophysitis, inflammatorybowel disease (Crohn's disease and ulcerative colitis), inflammatorypelvic disease, irritable bowel syndrome, Kawasaki disease, LPS-inducedendotoxic shock, meningitis, multiple sclerosis, myocarditis, myastheniagravis, mycosis fungoides, myositis, nephritis, osteomyelitis,pancreatitis, Parkinson's disease, pericarditis, pernicious anemia,pneumonitis, primary biliary sclerosing cholangitis, polyarteritisnodosa, psoriasis, retinitis, scleritis, scleracierma, scleroderma,sinusitis, Sjogren's disease, sepsis, septic shock, sunburn, systemiclupus erythematosus, tissue graft rejection, thyroiditis, type Idiabetes, Takayasu's arteritis, urethritis, uveitis, vasculitis,vasculitis including giant cell arteritis, vasculitis with organinvolvement such as glomerulonephritis, vitiligo, Waldenstrommacroglobulinemia and Wegener's granulomatosis.

The diseases and conditions that can be treated with the solid formprovided herein also include diseases and conditions which involveinflammatory responses to infections with bacteria, viruses, fungi,parasites or their toxins, such as sepsis, sepsis syndrome, septicshock, endotoxaemia, systemic inflammatory response syndrome (SIRS),multi-organ dysfunction syndrome, toxic shock syndrome, acute lunginjury, ARDS (adult respiratory distress syndrome), acute renal failure,fulminant hepatitis, burns, acute pancreatitis, post-surgical syndromes,sarcoidosis, Herxheimer reactions, encephalitis, myelitis, meningitis,malaria, SIRS associated with viral infections such as influenza, herpeszoster, herpes simplex and coronavirus.

The diseases for which the solid form provided herein are indicated alsoinclude diseases associated with a systemic inflammatory responsesyndrome, such as sepsis, burns, pancreatitis, major trauma, hemorrhageand ischemia. The solid form provided herein can be administered toreduce the incidence of: SIRS, the onset of shock, multi-organdysfunction syndrome, which includes the onset of acute lung injury,ARDS, acute renal, hepatic, cardiac and gastro-intestinal injury andmortality. For example, the compounds of the invention can beadministered prior to surgical or other procedures associated with ahigh risk of sepsis, hemorrhage, extensive tissue damage, SIRS or MODS.

Other diseases that can be treated with the solid form provided hereininclude viral infections. Examples of viral infections that can betreated include Epstein-Barr virus, hepatitis B virus, hepatitis Cvirus, herpes virus, human immunodeficiency virus, human papillomavirus, adenovirus, poxvirus and other episome-based DNA viruses. Thecompounds can therefore be used to treat disease and conditions such asherpes simplex infections and reactivations, cold sores, herpes zosterinfections and reactivations, chickenpox, shingles, human papillomavirus, cervical neoplasia, adenovirus infections, including acuterespiratory disease, and poxvirus infections such as cowpox and smallpoxand African swine fever virus. In one particular embodiment the solidform provided herein is indicated for the treatment of human papillomavirus infections of skin or cervical epithelia.

The diseases and conditions that can be treated with the solid formprovided herein also include conditions that are associated withischemia-reperfusion injury. Examples of such conditions include, butare not limited to conditions such as myocardial infarction,cerebrovascular ischemia (stroke), acute coronary syndromes, renalreperfusion injury, organ transplantation, coronary artery bypassgrafting, cardio-pulmonary bypass procedures and pulmonary, renal,hepatic, gastro-intestinal or peripheral limb embolism.

The solid form provided herein is also useful in the treatment ofdisorders of lipid metabolism via the regulation of APO-A1 such ashypercholesterolemia, atherosclerosis and Alzheimer's disease.

The solid form provided herein can also be used for the treatment offibrotic conditions such as idiopathic pulmonary fibrosis, renalfibrosis, post-operative stricture, keloid formation, scleroderma andcardiac fibrosis.

The solid form of provided herein can also be used to treatophthalmological indications such as dry eye.

As used herein, the term “contacting” refers to the bringing together ofindicated moieties in an in vitro system or an in vivo system. Forexample, “contacting” a BET protein with a compound described herein(e.g., the solid form provided herein) includes the administration ofthe compound to an individual or patient, such as a human, having a BETprotein, as well as, for example, introducing a compound of theinvention into a sample containing a cellular or purified preparationcontaining the BET protein.

As used herein, the term “individual” or “patient,” usedinterchangeably, refers to any animal, including mammals, preferablymice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep,horses, or primates, and most preferably humans.

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response that is being sought in a tissue,system, animal, individual or human by a researcher, veterinarian,medical doctor or other clinician.

As used herein, the term “treating” or “treatment” refers to one or moreof (1) inhibiting the disease; for example, inhibiting a disease,condition or disorder in an individual who is experiencing or displayingthe pathology or symptomatology of the disease, condition or disorder(i.e., arresting further development of the pathology and/orsymptomatology); and (2) ameliorating the disease; for example,ameliorating a disease, condition or disorder in an individual who isexperiencing or displaying the pathology or symptomatology of thedisease, condition or disorder (i.e., reversing the pathology and/orsymptomatology) such as decreasing the severity of disease.

As used herein, the term “preventing” or “prevention” refers topreventing a disease, condition or disorder in an individual who may bepredisposed to the disease, condition or disorder but does not yetexperience or display the pathology or symptomatology of the disease.

Combination Therapies

The solid form provided herein can be used in combination treatmentswhere the solid form of Compound 1-(S) described herein is administeredin conjunction with other treatments such as the administration of oneor more additional therapeutic agents. The additional therapeutic agentsare typically those which are normally used to treat the particularcondition to be treated. The additional therapeutic agents can include,e.g., chemotherapeutics, anti-inflammatory agents, steroids,immunosuppressants, as well as Bcr-Abl, Flt-3, RAF, FAK, and JAK kinaseinhibitors for treatment of BET protein-associated diseases, disordersor conditions. The one or more additional pharmaceutical agents can beadministered to a patient simultaneously or sequentially.

In some embodiments, the solid form provided herein can be used incombination with a therapeutic agent that targets an epigeneticregulator. Examples of epigenetic regulators include the histone lysinemethyltransferases, histone arginine methyl transferases, histonedemethylases, histone deacetylases, histone acetylases, and DNAmethyltransferases. Histone deacetylase inhibitors include, e.g.,vorinostat.

For treating cancer and other proliferative diseases, the compounds ofthe invention can be used in combination with chemotherapeutic agents,or other anti-proliferative agents. The compounds of the invention canalso be used in combination with medical therapy such as surgery orradiotherapy, e.g., gamma-radiation, neutron beam radiotherapy, electronbeam radiotherapy, proton therapy, brachytherapy, and systemicradioactive isotopes. Examples of suitable chemotherapeutic agentsinclude any of: abarelix, aldesleukin, alemtuzumab, alitretinoin,allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase,azacitidine, bevacizumab, bexarotene, bleomycin, bortezombi, bortezomib,busulfan intravenous, busulfan oral, calusterone, capecitabine,carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine,clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin,dalteparin sodium, dasatinib, daunorubicin, decitabine, denileukin,denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolonepropionate, eculizumab, epirubicin, erlotinib, estramustine, etoposidephosphate, etoposide, exemestane, filgrastim, floxuridine, fludarabine,fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumabozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan,idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a,irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leucovorin,leuprolide acetate, levamisole, lomustine, meclorethamine, megestrolacetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycinC, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine,nofetumomab, oxaliplatin, paclitaxel, pamidronate, panitumumab,pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatin,pipobroman, plicamycin, procarbazine, quinacrine, rasburicase,rituximab, ruxolitinib, sorafenib, streptozocin, sunitinib, sunitinibmaleate, tamoxifen, temozolomide, teniposide, testolactone, thalidomide,thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab,tretinoin, uracil mustard, valrubicin, vinblastine, vincristine,vinorelbine, vorinostat, and zoledronate.

For treating autoimmune or inflammatory conditions, the solid formprovided herein can be administered in combination with a corticosteroidsuch as triamcinolone, dexamethasone, fluocinolone, cortisone,prednisolone, or flumetholone.

For treating autoimmune or inflammatory conditions, the solid formprovided herein can be administered in combination with an immunesuppressant such as fluocinolone acetonide (Retisert®), rimexolone(AL-2178, Vexol, Alcon), or cyclosporine (Restasis®). For treatingautoimmune or inflammatory conditions, the solid form provided hereincan be administered in combination with one or more additional agentsselected from Dehydrex™ (Holles Labs), Civamide (Opko), sodiumhyaluronate (Vismed, Lantibio/TRB Chemedia), cyclosporine (ST-603,Sirion Therapeutics), ARG101(T) (testosterone, Argentis), AGR1012(P)(Argentis), ecabet sodium (Senju-Ista), gefarnate (Santen),15-(s)-hydroxyeicosatetraenoic acid (15(S)-HETE), cevilemine,doxycycline (ALTY-0501, Alacrity), minocycline, iDestrin™ (NP50301,Nascent Pharmaceuticals), cyclosporine A (Nova22007, Novagali),oxytetracycline (Duramycin, MOLI1901, Lantibio), CF101(2S,3S,4R,5R)-3,4-dihydroxy-5-[6-[(3-iodophenyl)methylamino]purin-9-yl]-N-methyl-oxolane-2-carbamyl,Can-Fite Biopharma), voclosporin (LX212 or LX214, Lux Biosciences),ARG103 (Agentis), RX-10045 (synthetic resolvin analog, Resolvyx), DYN15(Dyanmis Therapeutics), rivoglitazone (DE011, Daiichi Sanko), TB4(RegeneRx), OPH-01 (Ophtalmis Monaco), PCS101 (Pericor Science), REV1-31(Evolutec), Lacritin (Senju), rebamipide (Otsuka-Novartis), OT-551(Othera), PAI-2 (University of Pennsylvania and Temple University),pilocarpine, tacrolimus, pimecrolimus (AMS981, Novartis), loteprednoletabonate, rituximab, diquafosol tetrasodium (INS365, Inspire), KLS-0611(Kissei Pharmaceuticals), dehydroepiandrosterone, anakinra, efalizumab,mycophenolate sodium, etanercept (Embrel®), hydroxychloroquine, NGX267(TorreyPines Therapeutics), or thalidomide.

In some embodiments, the solid form provided herein can be administeredin combination with one or more agents selected from an antibiotic,antiviral, antifungal, anesthetic, anti-inflammatory agents includingsteroidal and non-steroidal anti-inflammatories, and anti-allergicagents. Examples of suitable medicaments include aminoglycosides such asamikacin, gentamycin, tobramycin, streptomycin, netilmycin, andkanamycin; fluoroquinolones such as ciprofloxacin, norfloxacin,ofloxacin, trovafloxacin, lomefloxacin, levofloxacin, and enoxacin;naphthyridine; sulfonamides; polymyxin; chloramphenicol; neomycin;paramomycin; colistimethate; bacitracin; vancomycin; tetracyclines;rifampin and its derivatives (“rifampins”); cycloserine; beta-lactams;cephalosporins; amphotericins; fluconazole; flucytosine; natamycin;miconazole; ketoconazole; corticosteroids; diclofenac; flurbiprofen;ketorolac; suprofen; cromolyn; lodoxamide; levocabastin; naphazoline;antazoline; pheniramine; or azalide antibiotic.

Other examples of agents, one or more of which the solid form providedherein may also be combined with include: a treatment for Alzheimer'sDisease such as donepezil and rivastigmine; a treatment for Parkinson'sDisease such as L-DOPA/carbidopa, entacapone, ropinirole, pramipexole,bromocriptine, pergolide, trihexyphenidyl, and amantadine; an agent fortreating multiple sclerosis (MS) such as beta interferon (e.g., Avonex®and Rebif®), glatiramer acetate, and mitoxantrone; a treatment forasthma such as albuterol and montelukast; an agent for treatingschizophrenia such as zyprexa, risperdal, seroquel, and haloperidol; ananti-inflammatory agent such as a corticosteroid, such as dexamethasoneor prednisone, a TNF blocker, IL-1 RA, azathioprine, cyclophosphamide,and sulfasalazine; an immunomodulatory agent, includingimmunosuppressive agents, such as cyclosporin, tacrolimus, rapamycin,mycophenolate mofetil, an interferon, a corticosteroid,cyclophosphamide, azathioprine, and sulfasalazine; a neurotrophic factorsuch as an acetylcholinesterase inhibitor, an MAO inhibitor, aninterferon, an anti-convulsant, an ion channel blocker, riluzole, or ananti-Parkinson's agent; an agent for treating cardiovascular diseasesuch as a beta-blocker, an ACE inhibitor, a diuretic, a nitrate, acalcium channel blocker, or a statin; an agent for treating liverdisease such as a corticosteroid, cholestyramine, an interferon, and ananti-viral agent; an agent for treating blood disorders such as acorticosteroid, an anti-leukemic agent, or a growth factor; or an agentfor treating immunodeficiency disorders such as gamma globulin.

In some embodiments, the solid form provided herein can be used incombination with one or more therapeutic agents selected from: Januskinase inhibitors (e.g., ruxolitinib, tofacitinib, baricitinib, CYT387,GLPG0634, lestaurtinib, pacritinib, TG101348), Pim kinase inhibitors,PI3 kinase inhibitors (including PI3K-delta selective and broad spectrumPI3K inhibitors), MEK inhibitors, cyclin dependent kinase inhibitors,b-RAF inhibitors, mTOR inhibitors, proteasome inhibitors (e.g.,bortezomib, carfilzomib), HDAC-inhibitors (e.g., panobinostat,vorinostat), DNA methyl transferase inhibitors, dexamethasone,melphalan, and immunomodulators such as lenolidomide and pomalidomide.In some embodiments, the Janus kinase inhibitor is selective for JAK1.In some embodiments, the Janus kinase inhibitor is selective for JAK1and JAK2.

In some embodiments, the solid form provided herein can be used incombination with one or more immune checkpoint inhibitors. Exemplaryimmune checkpoint inhibitors include inhibitors against immunecheckpoint molecules such as CD27, CD28, CD40, CD122, OX40, GITR, CD137,ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, indoleamine 2,3-dioxygenase(IDO), LAG3, TIM3, VISTA, PD-1, PD-1 and PD-L2. In some embodiments, thesolid form provided herein can be used in combination with one or moreagents selected from KIR inhibitors, TIGIT inhibitors, LAIR1 inhibitors,CD160 inhibitors, 2B4 inhibitors and TGFR beta inhibitors.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of PD-1, e.g., an anti-PD-1 monoclonal antibody. In someembodiments, the anti-PD-1 monoclonal antibody is nivolumab,pembrolizumab (also known as MK-3475), pidilizumab, SHR-1210, orAMP-224. In some embodiments, the anti-PD-1 monoclonal antibody isnivolumab or pembrolizumab.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of PD-L1, e.g., an anti-PD-L1 monoclonal antibody. In someembodiments, the anti-PD-L1 monoclonal antibody is BMS-935559, MEDI4736,MPDL3280A (also known as RG7446), or MSB0010718C. In some embodiments,the anti-PD-L1 monoclonal antibody is MPDL3280A or MEDI4736.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of CTLA-4, e.g., an anti-CTLA-4 antibody. In someembodiments, the anti-CTLA-4 antibody is ipilimumab.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of LAG3, e.g., an anti-LAG3 antibody. In some embodiments,the anti-LAG3 antibody is BMS-986016.

Formulation, Dosage Forms, and Administration

When employed as pharmaceuticals, the solid form provided herein can beadministered in the form of pharmaceutical compositions. In someembodiments, the present invention provides a pharmaceutical compositioncomprising the solid form provided herein and at least onepharmaceutically acceptable carrier. These compositions can be preparedin a manner well known in the pharmaceutical art, and can beadministered by a variety of routes, depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including transdermal, epidermal,ophthalmic and to mucous membranes including intranasal, vaginal andrectal delivery), pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal orintranasal), oral or parenteral. Parenteral administration includesintravenous, intra-arterial, subcutaneous, intraperitoneal intramuscularor injection or infusion; or intracranial, e.g., intrathecal orintraventricular, administration. Parenteral administration can be inthe form of a single bolus dose, or may be, for example, by a continuousperfusion pump. Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, the solid form provided herein, in combinationwith one or more pharmaceutically acceptable carriers (excipients). Insome embodiments, the present invention provides a solid oral dosageform comprising the solid form provided herein. In some embodiments, thedosage form is in the form of a pill, tablet, or capsule. In someembodiments, the composition is suitable for topical administration. Inmaking the compositions of the invention, the active ingredient istypically mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier in the form of, for example, a capsule, sachet,paper, or other container. When the excipient serves as a diluent, itcan be a solid, semi-solid, or liquid material, which acts as a vehicle,carrier or medium for the active ingredient. Thus, the compositions canbe in the form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments containing, for example, up to 10% byweight of the active compound, soft and hard gelatin capsules,suppositories, sterile injectable solutions, and sterile packagedpowders.

The compositions can be formulated as a solution or a suspension.Suitable excipients include methyl cellulose (MC), citrate, andD-α-Tocopherol polyethylene glycol 1000 succinate or Vitamin Epolyethylene glycol succinate (TPGS). In some embodiments, theconcentration of Compound 1-(S) is from about 0.1-5.0 mg/mL, about0.5-3.0 mg/mL, or about 0.5-2.5 mg/mL. The concentration of Compound1-(S) is about 0.5 mg/mL or about 2.5 mg/mL.

In some embodiments, the composition is a solution, comprising about 0.5mg/mL of Compound 1-(S) with about 0.5% MC in water. In someembodiments, the composition is a suspension, comprising about 2.5 mg/mLof Compound 1-(S) with about 0.5% MC in water. In some embodiments, thecomposition is a solution, comprising about 0.5 mg/mL of Compound 1-(S)with about 0.5% MC in about 50 mM citrate. In some embodiments, thecomposition is a suspension, comprising about 2.5 mg/mL of Compound1-(S) with about 0.5% MC in about 50 mM citrate. In some embodiments,the composition is a solution, comprising about 2.5 mg/mL of Compound1-(S) with about 0% TPGS in about 50 mM citrate.

In preparing a formulation, Compound 1-(S) can be milled to provide theappropriate particle size prior to combining with the other ingredients.Compound 1-(S) may be milled using known milling procedures such as wetmilling to obtain a particle size appropriate for tablet formation andfor other formulation types. Finely divided (nanoparticulate)preparations of Compound 1-(S) can be prepared by processes known in theart, e.g., see International App. No. WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 1,000 mg (1 g), more usually about 100mg to about 500 mg, of the solid form provided herein. The term “unitdosage forms” refers to physically discrete units suitable as unitarydosages for human subjects and other mammals, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical excipient.

In some embodiments, the compositions of the invention contain fromabout 5 mg to about 50 mg of the solid form provided herein. One havingordinary skill in the art will appreciate that this embodies Compound1-(S) and compositions thereof containing about 5 mg to about 10 mg,about 10 mg to about 15 mg, about 15 mg to about 20 mg, about 20 mg toabout 25 mg, about 25 mg to about 30 mg, about 30 mg to about 35 mg,about 35 mg to about 40 mg, about 40 mg to about 45 mg, or about 45 mgto about 50 mg of Compound 1-(S).

In some embodiments, the compositions of the invention contain fromabout 50 mg to about 500 mg of the solid form provided herein. Onehaving ordinary skill in the art will appreciate that this embodiesCompound 1-(S) and compositions thereof containing about 50 mg to about100 mg, about 100 mg to about 150 mg, about 150 mg to about 200 mg,about 200 mg to about 250 mg, about 250 mg to about 300 mg, about 350 mgto about 400 mg, or about 450 mg to about 500 mg of Compound 1-(S).

In some embodiments, the compositions of the invention contain fromabout 500 mg to about 1,000 mg of the solid form provided herein. Onehaving ordinary skill in the art will appreciate that this embodiesCompound 1-(S) and compositions thereof containing about 500 mg to about550 mg, about 550 mg to about 600 mg, about 600 mg to about 650 mg,about 650 mg to about 700 mg, about 700 mg to about 750 mg, about 750 mgto about 800 mg, about 800 mg to about 850 mg, about 850 mg to about 900mg, about 900 mg to about 950 mg, or about 950 mg to about 1,000 mg ofCompound 1-(S).

The active compound may be effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, Compound 1-(S) ismixed with a pharmaceutical excipient to form a solid preformulationcomposition containing a homogeneous mixture of Compound 1-(S). Whenreferring to these preformulation compositions as homogeneous, Compound1-(S) is typically dispersed evenly throughout the composition so thatthe composition can be readily subdivided into equally effective unitdosage forms such as tablets, pills and capsules. This solidpreformulation is then subdivided into unit dosage forms of the typedescribed above containing from, for example, about 0.1 to about 1000 mgof Compound 1-(S).

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which Compound 1-(S) and compositions thereof can beincorporated for administration orally or by injection include aqueoussolutions, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil, or peanut oil, as well as elixirs and similarpharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions can be nebulized by use of inert gases. Nebulized solutionsmay be breathed directly from the nebulizing device or the nebulizingdevice can be attached to a face masks tent, or intermittent positivepressure breathing machine. Solution, suspension, or powder compositionscan be administered orally or nasally from devices which deliver theformulation in an appropriate manner.

Topical formulations can contain one or more conventional carriers. Insome embodiments, ointments can contain water and one or morehydrophobic carriers selected from, for example, liquid paraffin,polyoxyethylene alkyl ether, propylene glycol, white vaseline, and thelike. Carrier compositions of creams can be based on water incombination with glycerol and one or more other components, e.g.,glycerinemonostearate, PEG-glycerinemonostearate and cetylstearylalcohol. Gels can be formulated using isopropyl alcohol and water,suitably in combination with other components such as, for example,glycerol, hydroxyethyl cellulose, and the like. In some embodiments,topical formulations contain at least about 0.1, at least about 0.25, atleast about 0.5, at least about 1, at least about 2, or at least about 5wt % of Compound 1-(S). The topical formulations can be suitablypackaged in tubes of, for example, 100 g which are optionally associatedwith instructions for the treatment of the select indication, e.g.,psoriasis or other skin condition.

The amount of Compound 1-(S) and compositions thereof administered to apatient will vary depending upon what is being administered, the purposeof the administration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. It will be understood that useof certain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of Compound 1-(S) can vary according to, forexample, the particular use for which the treatment is made, the mannerof administration of the compound, the health and condition of thepatient, and the judgment of the prescribing physician. The proportionor concentration of Compound 1-(S) in a pharmaceutical composition canvary depending upon a number of factors including dosage, chemicalcharacteristics (e.g., hydrophobicity), and the route of administration.For example, Compound 1-(S) can be provided in an aqueous physiologicalbuffer solution containing about 0.1 to about 10% w/v of the compoundfor parenteral administration. Some typical dose ranges are from about 1μg/kg to about 1 g/kg of body weight per day. In some embodiments, thedose range is from about 0.01 mg/kg to about 100 mg/kg of body weightper day. The dosage is likely to depend on such variables as the typeand extent of progression of the disease or disorder, the overall healthstatus of the particular patient, the relative biological efficacy ofthe compound selected, formulation of the excipient, and its route ofadministration. Effective doses can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

The compositions of the invention can further include one or moreadditional pharmaceutical agents such as a chemotherapeutic, steroid,anti-inflammatory compound, or immunosuppressant, examples of which arelisted hereinabove.

Kits

The present invention also includes pharmaceutical kits useful, forexample, in the treatment or prevention of BET protein-associateddiseases or disorders, such as cancer, which include one or morecontainers containing a pharmaceutical composition comprising atherapeutically effective amount of the solid form provided herein, orany of the embodiments thereof. Such kits can further include, ifdesired, one or more of various conventional pharmaceutical kitcomponents, such as, for example, containers with one or morepharmaceutically acceptable carriers, additional containers, etc., aswill be readily apparent to those skilled in the art. Instructions,either as inserts or as labels, indicating quantities of the componentsto be administered, guidelines for administration, and/or guidelines formixing the components, can also be included in the kit.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of non-criticalparameters which can be changed or modified to yield essentially thesame results.

EXAMPLES Example 1 Preparation of Amorphous Solid Form of Compound 1-(S)

Preparation of Starting Material 2-Bromo-1-Pyridin-2-YlethanoneHydrobromide

Bromine (Br₂, 198 g, 1240 mmol) dissolved in acetic acid (100 mL) wasadded slowly to a mixture of 1-(pyridin-2-yl)ethanone (150 g, 1238 mmol)in acetic acid (1500 mL) at rt. The red reaction solution was heated to105° C. for 1 hour to give an off-white slurry. The mixture was stirredfor 1 hour at rt, cooled in a water bath (caution acetic acid freezes),filtered, then the solids were washed with acetic acid (150 mL) andethyl acetate (250 mL). The solid was suspended in acetic acid (1500 mL)and heated to 105° C. for 1 h (note: material did not completelydissolve). The reaction was allowed to cool to rt in a water bath. Theprecipitate was filtered, washed with acetic acid 100 mL and ethylacetate 300 mL, and dried under house vacuum at rt to give crude2-bromo-1-pyridin-2-ylethanone hydrobromide (305 g, 87%) as an off-whitepowder, which was used in the subsequent step without furtherpurification.

Step 1. 5-nitro-3-(pyridin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-3-ol(1x)

2-Bromo-1-pyridin-2-ylethanone hydrobromide (135 g, 480 mmol) wassuspended in acetonitrile (1700 mL) at rt and potassium carbonate (135g, 977 mmol) was added. The reaction was stirred for 1 h and then washeated to 60° C. and 2-amino-3-nitrophenol (67.5 g, 438 mmol) was addedslowly portionwise as a solid over 30 minutes. The reaction became adark red slurry. This mixture was stirred at 60° C. for an additional 1h. The reaction was diluted with water (6500 mL) and stirred vigorouslyfor 1 h to give a precipitate. The solids were collected and washed withwater and dried to give the product as a dark brown solid 134 g. Thesolid was suspended in ethyl acetate (2500 mL) and heated to 75° C. inan oil bath to dissolve the product. To the reaction mixture was addedactivated charcoal (40 g). The mixture was stirred for 30 minutes andfiltered hot through celite to give a yellow-red solution. The solutionwas concentrated in vacuo to give a crystalline residue. This wastriturated with ethyl ether, filtered and the solids were washed withethyl ether to give the crude desire product,5-nitro-3-(pyridin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-3-ol (1x,100 g, 83%) as a yellow orange crystalline solid. The mother liquor wasconcentrated to give an additional 10.0 g of the crude desired productas a yellow orange solid. The crude desired product was directlyutilized in the subsequent reactions without further purification.

Step 2. 3-(Pyridin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-5-amine (2x)

A Parr reactor (18 L) was charged with5-nitro-3-(pyridin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-3-ol (1x,1000 g, 3.918 mol), methanol (7 L) and 10% palladium on carbon (97 g,50% wet) at ambient temperature. After a standard nitrogen purge, thevessel was charged with hydrogen (65 psi) and stirred for 16 hoursmaintaining the internal temperature between 20-25° C. The vessel wasrecharged with hydrogen 8 times. Once the reaction was complete, thevessel was purged again with nitrogen and the resultant dark solutionwas filtered through a pad of celite (300 g). The celite filter cake waswashed with methanol (6 L) and the combined filtrates were concentratedunder reduced pressure. Toluene (8 L) was added and evaporated underreduced pressure to remove water. The residue was purified over silicagel (SiO₂, 2 Kg), eluting with a gradient of 50 to 100% ethyl acetate inn-heptane. The solids were triturated with methyl tert-butyl ether(MTBE, 1 L) and filtered to give the first crop of the desired product(2x, 447 g) as off-white to yellow solids. The filtrates werere-chromatographed and triturated with MTBE to give the second crop ofthe desired product (2x, 145 g) as off-white to yellow solids. The totalamount of desired product (2x) isolated was 592 g (66.5% yield, 99%purity). ¹H NMR (300 MHz, DMSO-d₆) δ 8.60-8.56 (m, 1H), 7.83-7.76 (m,1H), 7.5-7.47 (m, 1H), 7.34-7.29 (m, 1H), 6.42-6.36 (m, 1H), 6.28-6.24(m, 1H), 6.10-6.06 (m, 1H), 5.15-5.13 (m, 1H) 4.69 (s, 2H), 4.58-4.54(m, 1H), 4.29-4.24 (m, 1H), 4.14-4.08 (m, 1H); C₁₃H₁₃N₃O (MW: 227.26),LCMS (EI) m/e 228 (M⁺+H).

Step 3.4-Pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(3x)

A suspension of3-(pyridin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-5-amine (2x, 10 g,0.044 mole, 1.0 equiv) in ethyl acetate (120 mL) was heated to 40-50° C.and stirred at 40-50° C. until a solution was obtained. The resultingsolution was gradually cooled down to 15-30° C. beforeN,N-carbonyldiimidazole (CDI, 8.55 g, 0.0528 mole, 1.2 equiv) was added.The resulting reaction mixture was stirred at 15-40° C. for at least 30minutes and the desired product (3x) precipitated out during thereaction. When HPLC showed the reaction was complete ((2x)<0.5%), thesolids were collected by filtration and washed sequentially with MTBE(20 mL), water (2×25 mL), and MTBE (20 mL). The wet cake was dried onthe filter for at least 3 hours, and then dried at 20-60° C. undervacuum to afford4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(3x, 9.92 g, 89% yield) as off-white solids. ¹H NMR (400 MHz, DMSO-d₆) δ10.91 (s, 1H), 8.51 (d, J=4.1 Hz, 1H), 7.74 (td, J=7.7, 1.8 Hz, 1H),7.29 (dd, J=6.8, 4.9 Hz, 1H), 7.01 (d, J=7.9 Hz, 1H), 6.85 (t, J=8.0 Hz,1H), 6.67 (d, J=7.7 Hz, 1H), 6.52 (d, J=8.1 Hz, 1H), 5.48 (s, 1H), 4.75(dd, J=11.5, 1.9 Hz, 1H), 4.39 (dd, J=11.5, 3.1 Hz, 1H) ppm; ¹³C NMR(101 MHz, DMSO-d₆) δ 157.24, 153.53, 150.04, 141.07, 137.90, 128.65,123.64, 121.75, 121.41, 118.18, 107.10, 103.33, 70.89, 54.55 ppm;C₁₄H₁₁N₃O₂ (MW: 253.26), LCMS (EI) m/e 254 (M⁺+H).

Step 4.7-Iodo-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(4x)

A solution of4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(3x, 25 g, 0.0987 mole) in N,N-dimethyl-acetamide (DMF, 300 ml) wasstirred at 15-30° C. for 15 minutes. N-Iodosuccinimide (NIS, 24.43 g,0.1086 mole, 1.10 equiv) was charged to the reaction mixture and theresulting reaction mixture was stirred at 15-30° C. for 12-60 hoursuntil the starting material4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(3x) was less than 10% by HPLC. Sulfuric acid (H₂SO₄, 1.58 mL, 0.003mole, 0.30 equiv) was then added while maintaining the batch at 15-30°C. The reaction mixture was stirred at 15-30° C. until starting material(3x) was less than 2%. The reaction mixture was then cooled to 5-15° C.before an 1 N aqueous sodium hydroxide (55.3 ml, 0.0553 mole, 0.56equiv) solution was added to adjust the reaction mixture pH to 4 to 7while maintaining the reaction temperature below 35° C. The resultingmixture was continuing to stir at 15-35° C. for at least 30 minutes andwater (50 mL) was added at 15-35° C. The resulting mixture was stirredat ambient temperature until a precipitate was observed. An additionalamount of water (250 mL) was added to the mixture and the resultingsuspension was stirred at ambient temperature for at least 1 hour beforethe solid was filtered. The filter cake was washed sequentially withwater (300 mL) and MTBE (175 mL). The wet cake was dried on the filterto afford7-iodo-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(4x, 31.4 g, 84% yield) as yellow to brown solids. ¹H NMR (400 MHz,DMSO-d₆) δ 11.03 (s, 1H), 8.50 (m, 1H), 7.76 (td, J=7.7, 1.8 Hz, 1H),7.31 (dd, J=6.8, 4.9 Hz, 1H), 7.22 (d, J=7.9 Hz, 1H), 7.10 (d, J=7.9 Hz,1H), 6.57 (t, J=8.2 Hz, 1H), 5.51 (s, 1H), 4.86 (dd, J=11.5, 1.8 Hz,1H), 4.46 (dd, J=11.5, 3.1 Hz, 1H) ppm; ¹³C NMR (101 MHz, DMSO-d₆) δ156.85, 153.09, 150.04, 140.99, 138.00, 129.81, 128.78, 123.77, 121.59,118.69, 105.93, 71.85, 70.86, 54.37 ppm; C₁₄H₁₀IN₃O₂ (MW: 379.15), LCMS(EI) m/e 380 (M⁺+H).

Step 5.7-(3,5-Dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 1)

1-Butanol (93.5 mL) was degassed by bubbling nitrogen for 5 minutes,then7-iodo-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(4x, 9.74 g, 0.026 mol), (3,5-dimethylisoxazol-4-yl)boronic acid (4.71g, 0.033 mole, 1.3 equiv), aqueous CsF solution (10.54 g in 16.6 mL ofH₂O, 0.069 mole, 2.7 equiv) and dichlorobis(p-dimethylaminophenylditbutylphosphine)palladium(II) (Pd-132) (0.091 g, 0.00013 mole,0.005 equiv) were charged to the reactor with nitrogen bubbling. Theresulting reaction mixture was further degassed by bubbling nitrogen for15 minutes before it was heated to 85-95° C. and stirred at 85-95° C.for 1-2 hour. When HPLC indicated reaction completion, the reactionmixture was cooled to 15-30° C. Then EtOAc (50 mL) and water (35 mL)were added, and the resulting mixture was stirred at ambient temperaturefor 30 minutes. The phases were separated, and the organic phase wasconcentrated under vacuum. The residue was transferred to a reactor withEtOAc and treated with an aqueous solution of L-cysteine. The resultingmixture was heated to 60-70° C. for 1 hour before being gradually cooledto 15-30° C. The two phases were separated and the organic phase wasfiltered through celite. The filter cake was washed with EtOAc. Theorganic phase was transferred to the reactor mixture with an additionalamount of aqueous L-cysteine solution and the resulting mixture washeated to 60-70° C. for 1 hour before being cooled down to 15-30° C. Thetwo phases were separated; the organic phase was filtered through celiteand the filter cake was washed with EtOAc. The organic phase was thenwashed with water and concentrated to afford7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 1, 8.95 g, 98.8% yield) as yellow to brown solids. ¹H NMR (400MHz, DMSO-d₆) δ 11.00 (s, 1H), 8.49 (m, 1H), 7.76 (td, J=7.7, 1.8 Hz,1H), 7.29 (ddd, J=7.5, 4.8, 0.9 Hz, 1H), 7.11 (d, J=8.0 Hz, 1H), 6.79(m, 2H), 5.52 (t, J=2.3 Hz, 1H), 4.76 (dd, J=11.4, 2.0 Hz, 1H), 4.43(dd, J=11.4, 3.1 Hz, 1H), 2.20 (s, 3H), 2.03 (s, 3H) ppm; ¹³C NMR (101MHz, DMSO-d₆) δ 166.13, 159.55, 157.12, 153.56, 149.97, 138.81, 137.90,128.45, 123.70, 123.31, 121.56, 118.66, 112.14, 108.91, 103.56, 71.07,54.37, 11.98, 10.90 ppm; C₁₉H₁₆N₄O₃ (MW: 348.36), LCMS (EI) m/e 349(M⁺+H).

Step 6.(4S)-7-(3,5-Dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 1-(S))

In an 8×50 mm Simulated Moving Bed (SMB) separation unit, the columnspacked with 20-micron Chiralpak AS chiral stationary phase (CSP) wereinstalled. The columns and the SMB system were then flushed withacetonitrile. A solution of7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 1, 5500 g) in acetonitrile was then loaded onto the chiralcolumns installed in the SMB unit. The chiral columns were then elutedwith acetonitrile. The fractions containing the desired Compound 1-(S)and meeting the chiral purity criterion as determined by chiral HPLCwere collected and combined for concentration under reduced pressure.After removal of acetonitrile, the crude desired product,(4S)-7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 1-(S), 2500 g, 45.5%) was obtained for further purification.The fractions containing the undesired (R)-enantiomer as determined bychiral HPLC, were collected and combined for concentration under reducedpressure to afford the crude undesired product, for other use. The crudeCompound 1-(S) obtained from the chiral separation was then dissolved inmethanol and the resulting methanol solution was loaded onto the samechiral columns installed in the same SMB unit. The chiral columns wereeluted with methanol. The fractions containing the desired product andmeeting the chemical purity criterion as determined by HPLC and chiralHPLC were collected, combined for concentration under reduced pressure.After removal methanol, the pure desired product,(4S)-7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 1-(S), 2300 g, 41.8% for two separations), was obtained aslight yellow oil, which was solidified under vacuum at ambienttemperature. This material was used directly in the subsequent processsteps to manufacture amorphous Compound 1-(S) free base drug substance.

Step 7.(4S)-7-(3,5-Dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(amorphous Compound 1-(S))

A suspension of(4S)-7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 1-(S), 42.86 g, 0.123 mole) in water (429 mL) was cooled to0-5° C. A 1 M aqueous NaOH solution (123 mL, 0.123 mole, 1.0 equiv) wasthen added to the suspension while maintaining the internal temperatureat 0-5° C. The reaction mixture (at pH 12-13) was then gradually warmedto 10-25° C. and stirred at 10-25° C. until a clear solution wasobtained. The solution was polish filtered, and rinsed through thefilter with a 0.25 M aqueous NaOH solution (20 mL, 0.005 mole, 0.04equiv). The filtered solution was then cooled to 0-5° C. before beingtreated with an 1 M aqueous HCl solution (128 mL, 0.128 mole, 1.04equiv) at 0-5° C. The final pH of the reaction mixture was 2-4. Thesolid product was gradually precipitated out during acidification andthe resulting suspension was gradually warmed to ambient temperature andstirred at ambient temperature for 1-2 hours. The solid was collected byfiltration and washed sequentially with purified water and n-heptane.The wet cake was dried by pulling vacuum on the filter for 3-10 hoursand further dried under vacuum at 30-40° C. to afford(4S)-7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(amorphous Compound 1-(S), 38.8 g, 90.6% yield) as white to off-whiteamorphous powder. ¹H NMR (400 MHz, DMSO-d₆) δ 11.00 (s, 1H), 8.49 (m,1H), 7.76 (td, J=7.7, 1.8 Hz, 1H), 7.29 (ddd, J=7.5, 4.8, 0.9 Hz, 1H),7.11 (d, J=8.0 Hz, 1H), 6.79 (m, 2H), 5.52 (t, J=2.3 Hz, 1H), 4.76 (dd,J=11.4, 2.0 Hz, 1H), 4.43 (dd, J=11.4, 3.1 Hz, 1H), 2.20 (s, 3H), 2.03(s, 3H) ppm; ¹³C NMR (101 MHz, DMSO-d₆) δ 166.13, 159.55, 157.12,153.56, 149.97, 138.81, 137.90, 128.45, 123.70, 123.31, 121.56, 118.66,112.14, 108.91, 103.56, 71.07, 54.37, 11.98, 10.90 ppm; C₁₉H₁₆N₄O₃ (MW:348.36), LCMS (EI) m/e 349 (M⁺+H).

Example 2 X-Ray Powder Diffraction (XRPD) of Amorphous Compound 1-(S)

Amorphous Compound 1-(S) was characterized by XRPD. The X-Ray PowerDiffraction (XRPD) was obtained from Rigaku MiniFlex X-ray PowderDiffractometer (XRPD). The general experimental procedures for XRPDwere: (1) X-ray radiation from copper at 1.054056 A with K_(β) filter;(2) X-ray power at 30 KV, 15 mA; and (3) the sample powder was dispersedon a zero-background sample holder. The general measurement conditionsfor XRPD were: Start Angle 3 degrees; Stop Angle 45 degrees; Sampling0.02 degrees; and Scan speed 2 degree/min. The XRPD pattern displays anamorphous halo as shown in FIG. 1.

Example 3 Differential Scanning Calorimetry (DSC) of Amorphous Compound1-(S)

Amorphous Compound 1-(S) was characterized by DSC. The DSC was obtainedfrom TA Instruments Differential Scanning calorimetry, Model Q200 withautosampler. The DSC instrument conditions were as follows: 30-350° C.at 10° C./min; Tzero aluminum sample pan and lid; and nitrogen gas flowat 50 mL/min. The DSC thermogram is shown in FIG. 2. The DSC thermogramrevealed one exothermic event at an onset temperature of 181.7° C. witha peak temperature of 213.4° C. which corresponds to the decompositionof the compound. The endothermic event below 100° C. is believed to bedue to dehydration. No melting point was observed due to the amorphousnature of the compound.

Example 4 Determination of Glass Transition Temperature of AmorphousCompound 1-(S)

The glass transition temperature (Tg) was determined to be 105.8° C. bymodulated DSC (MDSC) using TA Instruments Differential Scanningcalorimetry, Model Q2000 with autosampler. The MDSC instrumentconditions were as follows: modulation temperature amplitude: ±1° C.;modulation: 60 second; ramp rate: 2° C./min; temperature range: 10-150°C.; Tzero aluminum sample pan and lid; and nitrogen gas flow at 50mL/min. The MDSC thermogram is shown in FIG. 3.

Example 5 Stability of Amorphous Compound 1-(S)

Amorphous Compound 1-(S) has been found to be physically stable underelevated temperature and humidity. Table 1 indicates the conditions/timeto which the amorphous form was subjected and provides the measuredwater content (Karl-Fischer analysis) and purity (by HPLC/UV). TheHPLC/UV instrument was Agilent HPLC 1100 with an Agilent Zorbax SB-C18,3.5 μm, 4.6×150 mm column. The HPLC conditions were as follows: columntemperature: 40° C.; mobile phase A (MPA): 0.05% (v/v) trifluoroaceticacid (TFA) in water; mobile phase B (MPB): 0.05% (v/v) TFA inacetonitrile; and flow rate: 1 mL/min.

The gradient conditions were as follows:

Time (minute) % MPA % MPB 0 67 23 20 67 23 25 20 80 25.1 67 23 30 67 23Injection: 5 μL Detection: UV 220 nm, 254 nm

TABLE 1 Water Purity Purity Time content 220 nm 254 nm Solid FormConditions (weeks) (%) (%) (%) (XRPD) 50° C. 0 1.67 98.22 97.10Amorphous Open vial 1 0.69 98.12 97.11 Not measured 2 0.43 98.23 97.11Amorphous 40° C./75% RH 0 1.67 98.22 97.10 Amorphous Open vial 1 3.3998.22 97.10 Not measured 2 2.64 98.18 97.10 Amorphous

Example 6 Formulation of Amorphous Compound 1-(S)

Amorphous Compound 1-(S) was formulated as described in the table below.Table 2 indicates the target concentration, final pH, solubility, andsome observation of the resulting formulations.

TABLE 2 Target Concentration Final Solubility Vehicle (mg/mL) pH (mg/mL)Observations 0.5% MC in water 0.5 6.76 >0.5 solution 2.5 7.87 0.842suspension 0.5% MC in 0.5 3.00 >0.5 solution 50 mM citrate, pH 2.5 3.031.15 suspension 3.0 10% TPGS in 2.5 3.07 >2.5 solution 50 mM citrate, pH3.0 MC = methyl cellulose; TPGS = D-α-tocopherol polyethylene glycol1000 succinate or Vitamin E polyethylene glycol succinate.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference cited in the presentdisclosure, including all patent, patent applications, and publications,is incorporated herein by reference in its entirety.

1-39. (canceled)
 40. A method for preparing(4S)-7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 1-(S)), comprising: reacting7-iodo-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 4x) with (3,5-dimethylisoxazol-4-yl)boronic acid in thepresence of a palladium complex to afford7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 1); and separating the S enantiomer of Compound 1 using chiralcolumn chromatography to afford Compound 1-(S).
 41. A method forpreparing7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 1), comprising reacting7-iodo-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 4x) with (3,5-dimethylisoxazol-4-yl)boronic acid in thepresence of a palladium complex.
 42. The method of claim 40 wherein thereacting is carried out in the presence of an aqueous CsF solution andan organic solvent.
 43. The method of claim 40 wherein the palladiumcomplex is dichlorobis(p-dimethylaminophenylditbutylphosphine)palladium(II).
 44. The method of claim 42wherein the organic solvent is n-butanol.
 45. The method of claim 40wherein Compound 4x is prepared by reacting 4-pyridin-2-yl-4,5-dihydroimidazo[1,5, 4-de][1,4]benzoxazin-2(1H)-one (Compound 3x) withN-iodosuccinimide. 46-51. (canceled)
 52. The method of claim 45 whereinCompound 3x is prepared by reacting3-(pyridin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-5-amine (Compound2x) with N,N-carbonyldiimidazole in the presence of an organic solvent.53. The method of claim 52 wherein the organic solvent is ethyl acetate.54. The method of claim 52 wherein Compound 2x is prepared by reacting5-nitro-3-(pyridin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-3-ol(Compound 1x) with hydrogen in the presence of palladium on carbon andan organic solvent.
 55. The method of claim 54 wherein the organicsolvent is methanol.
 56. The method of claim 54 wherein Compound 1x isprepared by reacting 2-bromo-1-pyridin-2-ylethanone hydrobromide with2-amino-nitrophenol in the presence of a base and an organic solvent.57. (canceled)
 58. The method of claim 56 wherein the base is K₂CO₃. 59.The method of claim 56 wherein the organic solvent is acetonitrile. 60.A method for preparing Compound 1-(S), comprising 1) reacting2-bromo-1-pyridin-2-ylethanone hydrobromide with 2-amino-nitrophenol inthe presence of a base and an organic solvent to afford5-nitro-3-(pyridin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-3-ol(Compound 1x); 2) reacting Compound 1x with hydrogen in the presence ofpalladium on carbon and an organic solvent to afford3-(pyridin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-5-amine (Compound2x); 3) reacting Compound 2x with N,N-carbonyldiimidazole in thepresence of an organic solvent to afford4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 3x); 4) reacting Compound 3x with N-iodosuccinimide to afford7-iodo-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 4x); 5) reacting Compound 4x with(3,5-dimethylisoxazol-4-yl)boronic acid in the presence of a palladiumcomplex to afford7-(3,5-dimethylisoxazol-4-yl)-4-pyridin-2-yl-4,5-dihydroimidazo[1,5,4-de][1,4]benzoxazin-2(1H)-one(Compound 1); and 6) separating the S enantiomer of Compound 1 usingchiral column chromatography to afford Compound 1-(S).